2 Laboratory performance

2.1 Load data

Load data previously compiled from the database:

all_data <- read_tsv("data/all_data.tsv") %>%
    mutate(Taxon=factor(Taxon,levels=c("Amphibian",
                                       "Reptile",
                                       "Mammal",
                                       "Bird",
                                       "Control"))) %>%
    mutate(Extraction=factor(Extraction,levels=c("ZYMO",
                                                 "DREX",
                                                 "EHEX"))) %>%
    mutate(Species=factor(Species,levels=c("Calotriton asper",
                                           "Lissotriton helveticus",
                                           "Salamandra atra",
                                           "Chalcides striatus",
                                           "Natrix astreptophora",
                                           "Podarcis muralis",
                                           "Plecotus auritus",
                                           "Sciurus carolinensis",
                                           "Trichosurus vulpecula",
                                           "Geospizopsis unicolor",
                                           "Perisoreus infaustus",
                                           "Zonotrichia capensis",
                                           "Extraction control",
                                           "Library control")))

2.2 DNA yield

Total amount of DNA extracted from the 150 ul subset of the bead-beaten sample.

all_data %>%
    select(Extraction,extract,Taxon) %>%
    group_by(Taxon,Extraction) %>%
    summarise(value = sprintf("%.0f±%.0f", mean(extract), sd(extract))) %>%
    pivot_wider(names_from = Extraction, values_from = value) %>%
    tt(caption = "Mean and standard deviation of total DNA nanograms")

Mean and standard deviation of total DNA nanograms

Taxon	ZYMO	DREX	EHEX
Amphibian	471±347	482±546	1733±1145
Reptile	102±68	162±128	250±171
Mammal	445±394	265±214	904±672
Bird	73±127	13±12	54±45
Control	0±0	2±3	1±0

all_data %>%
    select(Library,Species,Extraction,extract,Taxon) %>%
    unique() %>%
    ggplot(aes(x=Extraction, y=extract, color=Species, group=Extraction))+ 
        geom_boxplot(outlier.shape = NA, fill="#f4f4f4", color="#8c8c8c") + 
        geom_jitter() + 
        scale_color_manual(values=vertebrate_colors) +
        facet_grid(. ~ Taxon, scales = "free") +
        theme_minimal() +
        labs(y="DNA yield (ng)",x="Extraction method")

all_data  %>%
    filter(Taxon != "Control") %>%
    lmerTest::lmer(extract ~ Extraction + (1 | Sample) + (1 | Species), data = ., REML = FALSE) %>%
    broom.mixed::tidy() %>%
    tt()

effect	group	term	estimate	std.error	statistic	df	p.value
fixed	NA	(Intercept)	160.36395	125.49705	1.2778304	16.14808	2.193741e-01
fixed	NA	ExtractionDREX	-39.65796	74.42689	-0.5328445	153.24421	5.949131e-01
fixed	NA	ExtractionEHEX	580.62175	74.78157	7.7642359	153.30986	1.088065e-12
ran_pars	Sample	sd__(Intercept)	164.00812	NA	NA	NA	NA
ran_pars	Species	sd__(Intercept)	374.85139	NA	NA	NA	NA
ran_pars	Residual	sd__Observation	400.80106	NA	NA	NA	NA

DNA yields vary across host taxa. - Amphibians and mammals yield the highest total amounts of DNA. - Birds yield the lowest amounts of DNA. - Controls yield barely quantifiable DNA. EHEX yields highest amounts of total DNA. - DNA recovery raks of extraction methods are maintained across taxa.

2.3 Library performance

Number of PCR cycles required for reaching the plateau phase of the indexing PCR. A higher number indicates a lower amount of amplifiable library template. When weighed by the input DNA, the required number of PCR cycles serves as an estimator of library performance.

all_data %>%
    group_by(Taxon,Extraction) %>%
    summarise(value = sprintf("%.1f±%.1f", mean(pcr), sd(pcr))) %>%
    pivot_wider(names_from = Extraction, values_from = value) %>%
    tt(caption = "Mean and standard deviation of optimal number of PCR cycles")

Mean and standard deviation of optimal number of PCR cycles

Taxon	ZYMO	DREX	EHEX
Amphibian	14.3±1.6	10.7±2.9	10.7±2.3
Reptile	11.7±4.0	10.3±3.5	12.0±3.3
Mammal	11.1±3.5	9.9±1.9	10.0±2.1
Bird	17.6±2.8	18.7±4.3	14.4±2.3
Control	20.0±2.8	19.8±0.5	22.0±4.2

all_data %>%
    select(Library,Species,Extraction,pcr,Taxon) %>%
    unique() %>%
    ggplot(aes(x=Extraction,y=pcr, color=Species, group=Extraction))+ 
        scale_y_reverse() +
        geom_boxplot(outlier.shape = NA, fill="#f4f4f4", color="#8c8c8c") + 
        geom_jitter() + 
        scale_color_manual(values=vertebrate_colors) +
        facet_grid(. ~ Taxon, scales = "free") +
        theme_minimal() +
        labs(y="Optimal number of PCR cycles",x="Extraction method")

all_data  %>%
    filter(Taxon != "Control") %>%
    lmerTest::lmer(pcr ~ Extraction + (1 | Sample) + (1 | Species), data = ., REML = FALSE) %>%
    broom.mixed::tidy() %>%
    tt()

effect	group	term	estimate	std.error	statistic	df	p.value
fixed	NA	(Intercept)	13.862035	0.8648942	16.027435	14.90821	8.328452e-11
fixed	NA	ExtractionDREX	-1.568966	0.5106532	-3.072468	150.76244	2.519836e-03
fixed	NA	ExtractionEHEX	-2.091261	0.5130831	-4.075872	150.84234	7.389947e-05
ran_pars	Sample	sd__(Intercept)	1.068964	NA	NA	NA	NA
ran_pars	Species	sd__(Intercept)	2.599229	NA	NA	NA	NA
ran_pars	Residual	sd__Observation	2.749952	NA	NA	NA	NA